CN102925589B - Establishment method of reverse transcription loop-mediated isothermal amplification (RT-LAMP) detection for Prunus necrotic ringspot virus (PNRSV) - Google Patents
Establishment method of reverse transcription loop-mediated isothermal amplification (RT-LAMP) detection for Prunus necrotic ringspot virus (PNRSV) Download PDFInfo
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Abstract
The invention discloses establishment of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) detection method for Prunus necrotic ringspot virus (PNRSV). According to the RT-LAMP detection method for the PNRSV, four specific primers are designed for six regions of a target gene PNRSV, a type of strand displacement deoxyribonucleic acid (DNA) polymerase is utilized at the constant temperature of around 65 DEG C, and efficient amplification of nucleic acids can be achieved in dozens of minutes. The four specific primers are used for identifying the six specific sequence regions of a target sequence, and therefore high specificity of amplification of LAMP is guaranteed. In the process of LAMP, thermal denaturation of template is not needed, temperature is cycled for a long time, the amplification is carried out under isothermal conditions, a waste of time due to temperature change is not caused, and the reaction speed can be increased by 30-50%. Based on whether visible white precipitate of magnesium pyrophosphate exists in a reaction tube, whether the nucleic acids are amplified can be easily judged. The RT-LAMP method for detection of the PNRSV is high in specificity, and the RT-LAMP method for detection of the PNRSV has the advantages of being rapid, high in efficiency, simple in instrument requirement, simple and convenient to operate, low in cost and easy to popularize and use at the grass-roots.
Description
Invention field
The invention belongs to biotechnology virus detection techniques field, specifically, the present invention is specifically related to the technical field of a kind of ring mediated isothermal amplification (LAMP) technology for detection Prunus necrotic ring spot virus (PNRSV).
Background technology
At present the Molecular Detection of viral nucleic acid is adopted more all kinds of technology of PCR-based reaction, as conventional PCR, nest-type PRC, real-time fluorescence PCR etc., not only need the plant and instrument that PCR instrument, electrophoresis apparatus, gel imaging instrument etc. are expensive, and schedule of operation very complicated, time are long, the technical requirements higher to testing staff, has limited using and promoting as fast diagnosis method greatly.
Ring mediated isothermal amplification (LAMP) is that Notomi etc. is in a kind of constant temperature nucleic acid amplification method of exploitation in 2000, have high specificity, simple to operate, do not need complicated instrument, the features such as naked eyes can directly be observed detected result, and its high efficiency, simplification, with low cost and sense cycle are short are very suitable for basic unit and on-the-spot use; This technology, for 6 sections of goal gene, designs 4 special primers and utilizes a kind of archaeal dna polymerase with strand displacement activity, at a certain temperature nucleic acid is carried out to isothermal duplication; Started to be applied to the detection of biological infective pathogen recent years.Before this, the existing report that is applied in the detections of pathogenic micro-organism such as streptococcus pneumoniae, west nile virus, SARS virus, tomato yellow mosaic virus, Chinese yam mosaic poison, mycoplasma pneumoniae and the aspects such as somatotype of dengue virus, a few days ago, there is not yet the report that utilizes LAMP technology for detection Prunus necrotic ring spot virus.
Prunus necrotic ring spot virus (PNRSV) is the dangerous Quarantine Objectss of two classes in the dangerous venereal disease of the inward Plant Quarantine of the < < People's Republic of China (PRC), worm, weed species > >, is also the Quarantine Objects of many national imports in the world.PNRSV belongs to Bromoviridae (Bromoviridae) Ilarvirus and belongs to (Ilarvirus), and plastochondria form such as is at the rotational symmetry polyhedron, and diameter is about 22-23nm, has three kinds of big or small particles.PNRSV is the RNA viruses of positive strand, and nucleic acid size is 8.056kb, for triad linear.PNRSV carries out long-distance communications by asexual propagation materials such as seed and nursery stocks, can also spread through bamboo telegraphs such as pollen, entomophila, grafting, mechanical inoculations.PNRSV can infect the multiple fruit trees such as almond, cherry, peach, Lee, apricot and west (sweet) melon, cotton, Sunflower Receptacle, Kidney bean, hops Deng21Ge section, 47 belong to 189 kind of plant, have host range extensively, infect that transmissibility is strong, harm is serious, quarantine and the feature such as prevention and control difficulty is large.Fruit tree is just with throughout one's life poison once infecting, increases the weight of year by year, endangers lastingly, cause blade downright bad ring spot and perforation, fruit deformity, quality declines, and rots to accelerate, and tree body sharply fails, withered ahead of time, generally can cause 30%～70% production loss, serious caused bark necrosis, cancerous swelling are until whole strain total crop failure, withered is a kind of destructive disease in production of fruit trees.The generation of this disease has at present spreaded all over Europe, Asia, Africa, South America and North America and countries and regions, Oceanian more than 40 temperate zones.At Chinese PNRSV, also only limiting at present indivedual areas occurs.
Summary of the invention
At present for the state of the art having no in prior art specially for ring mediated isothermal amplification (LAMP) technology for detection Prunus necrotic ring spot virus, the present invention aims to provide a kind of reverse transcription loop-mediated isothermal amplification detection method of setting up Prunus necrotic ring spot virus, carry out quick, special, sensitive, easy Site Detection, overcome the deficiency of prior art.
The present invention is achieved through the following technical solutions: the present invention is according to the conserved sequence of PNRSV isolate CP gene, designed PNRSV RT-LAMP Auele Specific Primer, determine reaction conditions and reagent concentration and be optimized, set up the RT-LAMP rapid detection system of PNRSV, this method is strong to the detection specificity of PNRSV, 2h can obtain detected result, has quick, efficient, easy feature.
The invention provides a kind of reverse transcription loop-mediated isothermal amplification detection method of setting up Prunus necrotic ring spot virus, comprise the following steps:
(1) provide a set of LAMP atopic primer sequence for detection of Prunus necrotic ring spot virus, concrete oligonucleotide sequence is:
F3：AATCATACCCACGCTGGTG
B3：AATTCGGGGAGGCACATTC
FIP：TTCGCAGCCCTTTGTTGAGCC-TTGCAAGAAGTGCCATCCG
BIP：TAGGGTTTCGAGCGGTGTAGGA-TCACGGTCCAAGTGGTCT
(2) provide the LAMP detection method of Prunus necrotic ring spot virus, i.e. preparation feedback template from detected sample first, and prepare LAMP reaction system, then by LAMP response procedures, reaction template is increased.
(3) finally according to the turbidity of reaction mixture, detected result is judged.
The present invention specifically provides a kind of reverse transcription loop-mediated isothermal amplification detection method of setting up Prunus necrotic ring spot virus, comprises the following steps:
(1) be designed for detect Prunus necrotic ring spot virus LAMP primer: according to the CP gene conserved sequence of the PNRSV isolate of announcing in prior art report and GenBank, utilize LAMP primer-design software Primer Explorer V4 design 1 cover Auele Specific Primer, comprise 1 pair of outer primer and 1 pair of inner primer, primer sequence following (5 '-3 '):
F3：AATCATACCCACGCTGGTG
B3：AATTCGGGGAGGCACATTC
FIP：TTCGCAGCCCTTTGTTGAGCC-TTGCAAGAAGTGCCATCCG
BIP：TAGGGTTTCGAGCGGTGTAGGA-TCACGGTCCAAGTGGTCT
(2) extraction of the total RNA of plant: get 0.1g fresh plant blade or young shoot is ground to powder in liquid nitrogen, adopt RNAplant plus Reagen test kit (Plant Genomic DNA Kit, TIANGEN) extract the total RNA of sample, extracting RNA product adds DEPC-ddH
2o dissolves, and-70 ℃ save backup.
(3) reverse transcription: the total RNA of plant extracting of take is template, and cDNA sequence is synthesized in reverse transcription.The reverse transcription system of 20 μ L is RNA template 2 μ L, the dNTP 1 μ L of 10mmol/L, the OligodT 1 μ L of 50 μ mol/L, DEPC-ddH
2o 10 μ L, process 5min at 65 ℃, place 5min on ice, add 5 * first-strand Buffer, 4 μ L, the M-MLV ThermoScript II 1 μ L of 200U/ μ L, the RNA enzyme inhibitors 1 μ L of 40U/ μ L; Reaction conditions: at 30 ℃, place 10min, 42 ℃ of insulation 1h, then in 95 ℃ of reaction 5min.
(4) preparation LAMP reaction system: reaction system is 27uL, the consumption of each component is: 10 * Buffer damping fluid 2.5uL, adopt the reverse transcription product cDNA 5.0uL of step (3), the dNTP6.0uL of 2.5mmol/L, the primers F 3 of 10 μ mol/L, each 0.5uL of B3, each 4.0uL of the primers F IP of 10 μ mol/L, BIP, the Betain 5.0uL of 100 μ mol/L, the MgSO of 100mmol/L
41.5uL, the Bst archaeal dna polymerase 1.0uL of 8U/ μ L arranges negative control simultaneously.
(5) by LAMP response procedures, reaction template is increased: LAMP response procedures: 95 ℃ of heating 5min; Chilling 3min on ice, then add 1.0 μ L Bst archaeal dna polymerases, 65 ℃ of water-bath 1.5h, 80 ℃ of deactivation 2min.
(6) detection of LAMP amplified production: increase and finish the variation of rear visual inspection reaction tubes turbidity by above-mentioned steps; To at the bottom of observation tube after amplified production high speed centrifugation, have or not magnesium pyrophosphate white precipitate, as occurred, white precipitate judges that detected result is positive, in sample, infect and have Prunus necrotic ring spot virus, on the contrary negative.
By implementing the concrete summary of the invention of the present invention, can reach following effect:
The present invention is directed to 4 special primers of 6 zone design of goal gene PNRSV, utilize a kind of strand displacement archaeal dna polymerase (Bst DNA polymerase) in 65 ℃ of left and right of isothermal, dozens of minutes can realize the efficient amplification of nucleic acid; Article 4, the identification in 6 of primer pair target sequence distinguished sequence regions, has guaranteed the high degree of specificity that LAMP increases; LAMP does not need the thermally denature of template, and long-time temperature cycle increases under isothermal condition, can, because temperature change causes waste of time, not make speed of response can improve 30-50%; According to having or not macroscopic magnesium pyrophosphate white precipitate in reaction tubes or add fluorescence dye SYBYGreen I in product, as the simple method that judges whether nucleic acid increases; This technology has started to be applied to the aspects such as diagnosis, Animal Sex evaluation and genetically modified food detection of Plant Quarantine, animal epidemic, has obtained gratifying achievements, shows wide application prospect, is specifically described below:
(1) high specificity.Special primer used is according to 6 different zones designs in PNRSV housing protein gene, and specificity surpasses conventional PCR.
(2) detect fast.From the extraction of RNA, to detecting about 2h of complete used time, and more than RT-PCR needs 5h, save detection time.
(3) instrument requires simple and easy.Water-bath or metal bath do not need regular-PCR PCR instrument, gel electrophoresis and imaging system used, as long as just can complete detection reaction.
(4) easy and simple to handle, result is obvious.Whole testing process does not relate to complex instrument or equipment, and the personnel of molecular biology mechanism of slightly having get final product complete operation; Detected result is clear, directly with eyes, observes and is just judged.
(5) to human and environment safety.Avoided conventional PCR testing process to use the poisonous pharmaceutical chemicalss such as ethidium bromide, all safer to human and environment.
(6) low cost.LAMP detects total cost and is significantly less than existing PCR detection method.
In a word, the method has the specificity higher than the regular-PCR detection method of reporting in prior art, the advantage such as simple and easy to do, quick.
Embodiment
Below, for embodiment, the present invention is described, still, the present invention is not limited to following embodiment.In addition, in following explanation, if no special instructions, % all refers to mass percent.
Plant and instrument: plant total RNA extraction reagent box, dNTPs is purchased from Beijing Tian Gen biochemical technology company limited.Reverse transcription test kit (BioTeke super RT Kit) is purchased from Beijing hundred Tyke Bioisystech Co., Ltd.Bst archaeal dna polymerase large fragment is Beijing Niu Yinglun NEB Bioisystech Co., Ltd product.
Test materials: take that to adopt the almond blade that contains PNRSV that DASELISA immunoabsorption (DAS-ELISA) detects be material, the negative contrast of healthy almond blade.
All reagent of selecting in the present invention and instrument are all well known selecting, but do not limit enforcement of the present invention, and other reagent more well known in the art and equipment are all applicable to the enforcement of the following embodiment of the present invention.
Embodiment mono-: the reverse transcription loop-mediated isothermal amplification detection method of setting up Prunus necrotic ring spot virus
The reverse transcription loop-mediated isothermal amplification detection method of setting up Prunus necrotic ring spot virus, comprises the following steps:
Be designed for detect Prunus necrotic ring spot virus LAMP Auele Specific Primer
According to the CP gene conserved sequence of the PNRSV isolate of announcing in prior art report and GenBank, utilize LAMP primer-design software Primer Explorer V4 design 1 cover Auele Specific Primer, comprise 1 pair of outer primer and 1 pair of inner primer, primer sequence following (5 '-3 '):
F3：AATCATACCCACGCTGGTG
B3：AATTCGGGGAGGCACATTC
FIP：TTCGCAGCCCTTTGTTGAGCC-TTGCAAGAAGTGCCATCCG
BIP：TAGGGTTTCGAGCGGTGTAGGA-TCACGGTCCAAGTGGTCT
(2) extraction of the total RNA of plant
Get the fresh susceptible almond blade of 0.1g or young shoot and in liquid nitrogen, be ground to powder, adopt RNAplantplus Reagen test kit (Plant Genomic DNA Kit, TIANGEN) to extract the total RNA of sample.Extracting RNA product adds DEPC-ddH
2o dissolves, and-70 ℃ save backup.With the negative contrast of healthy almond blade, and the known positive contrast of almond blade of having infected Prunus necrotic ring spot virus is set.
(3) reverse transcription
The total RNA of above-mentioned plant having extracted is template, and cDNA sequence is synthesized in reverse transcription.The reverse transcription system of 20 μ L is: RNA template 2 μ L, the dNTP 1 μ L of 10mmol/L, the OligodT 1 μ L of 50 μ mol/L, DEPC-ddH
2o 10 μ L, process 5min at 65 ℃, place 5min on ice, add 5 * first-strandBuffer, 4 μ L, the M-MLV ThermoScript II 1 μ L of 200U/ μ L, the RNA enzyme inhibitors 1 μ L of 40U/ μ L; Reaction conditions: at 30 ℃, place 10min, 42 ℃ of insulation 1h, then in 95 ℃ of reaction 5min.
(4) preparation LAMP reaction system:
Reaction system is 27uL, the consumption of each component is: 10 * Buffer damping fluid 2.5uL, according to the reverse transcription product cDNA5.0uL of step (3), the dNTP6.0uL of 2.5mmol/L, the primers F 3 of 10 μ mol/L, each 0.5uL of B3, each 4.0uL of the primers F IP of 10 μ mol/L, BIP, the Betain5.0uL of 100 μ mol/L, the MgSO of 100mmol/L
41.5uL, the Bst archaeal dna polymerase 1.0uL of 8U/ μ L arranges negative control simultaneously.
(5) by LAMP response procedures, reaction template is increased:
LAMP response procedures: 95 ℃ of heating 5min; Chilling 3min on ice, then add 1.0 μ L Bst archaeal dna polymerases, 65 ℃ of water-bath 1.5h, 80 ℃ of deactivation 2min.
(6) detection of LAMP amplified production: amplification finishes the variation of rear visual inspection reaction tubes turbidity.Magnesium pyrophosphate white precipitate will be had or not at the bottom of observation tube after amplified production high speed centrifugation.As all occurred white precipitate in detected sample and positive control reaction tubes, and in negative control reaction tubes without white precipitate, judge that detected result is positive, in sample, infect Prunus necrotic ring spot virus; As occurred white precipitate in detected example reaction pipe, and all not occurring white precipitate in positive control pipe, negative control reaction tubes, is false positive, in sample, does not infect Prunus necrotic ring spot virus; As all do not occurred white precipitate in detected sample, positive control pipe, negative control reaction tubes, negative, in sample, do not infect Prunus necrotic ring spot virus.
Embodiment bis-: the optimization of LAMP detection system
By three of the major effect of constructive system factor BstDNA polymerase concentration, cDNA add-on, reaction times length are tested, screened, each influence factor arranges gradient.Referring to table 1.
Table 1 Bst archaeal dna polymerase, cDNA add-on, reaction times gradient
1. the impact of reaction times length on RT-LAMP amplification
Get 10 identical sterilizing PCR pipes, wherein 5 pipes are made as negative control, and with the DEPC-ddH2O replacement cDNA template of equivalent, another 5 pipes, as positive reaction pipe, all by the LAMP reaction system of above-mentioned foundation, add respectively corresponding reagent and template in all pipes.After sealing and mixing, get respectively 1 negative control pipe and 1 positive reaction pipe under the suitableeest temperature of reaction condition, the 30min that increases respectively, 45min, 60min, 75min, 90min, 105min, finally reacts 2min with termination reaction at 80 ℃.
By test, show, along with the increase in reaction times, white precipitate increases gradually, starts to occur faint precipitation when the reaction times reaches 45min, and it is maximum that precipitation capacity reaches during the reaction times at 90min, and therefore, the suitable reaction times of LAMP is 90min.
The impact of 2.cDNA add-on on RT-LAMP amplification
Get 6 identical sterilizing PCR pipes, wherein 1 pipe is made as negative control, with the DEPC-ddH2O replacement cDNA template of equivalent; Another 5 pipes, as positive reaction pipe, add respectively 2 μ L, 3 μ L, and 4 μ L, 5 μ L, 6 μ L, the cDNA template of 8 μ L, all the other reagent all add respectively by the LAMP reaction system of above-mentioned foundation.After sealing and mixing, under the suitableeest temperature of reaction and optimal reaction time conditions, increase, finally at 80 ℃, react 2min with termination reaction.Under this test conditions, when reaching 5 μ L, cDNA add-on there is obvious white precipitate, when cDNA add-on is brought up to 6 μ L, white precipitate amount does not significantly increase.Therefore, the suitable add-on of cDNA is 5 μ L.
The impact of 3.Bst archaeal dna polymerase concentration on RT-LAMP amplification
Get 6 identical sterilizing PCR pipes, wherein 1 pipe is made as negative control, and with the DEPC-ddH2O replacement cDNA template of equivalent, another 5 pipes are as positive reaction pipe, add respectively 6U/ μ L, 8U/ μ L, 12U/ μ L, 18U/ μ L, 24U/ μ L, the BstDNA polysaccharase 1 μ L of 30U/ μ L, and by the LAMP reaction system of above-mentioned foundation, add respectively corresponding reagent and template.After sealing and mixing, under the suitableeest temperature of reaction and optimal reaction time conditions, increase, finally at 80 ℃, react 2min with termination reaction.Test-results shows, in BstDNA polymerase concentration is the scope of 6U-30U/ μ L, in the reaction tubes of 30U/ μ L, 24U/ μ L, all without precipitation, occur, during 18U/ μ L, start to occur precipitation, afterwards along with BstDNA polymerase concentration reduces, precipitation capacity increases, and when concentration is 8U/ μ L, precipitation capacity is maximum.Therefore, the suitable concentration of BstDNA polysaccharase is 8U/ μ L.
In sum, through optimizing the LAMP reaction system of detection Prunus necrotic virus later, be: 10 * Buffer damping fluid 2.5uL, cDNA template 5.0uL, the dNTP 6.0uL of 2.5mmol/L, the primers F 3 of 10 μ mol/L, each 0.5uL of B3, each 4.0uL of the primers F IP of 10 μ mol/L, BIP, the Betain 5.0uL of 100 μ mol/L, the MgSO of 100mmol/L
41.5uL, 8U/ μ L Bst archaeal dna polymerase 1uL.
Embodiment tri-: LAMP detection system specificity
Press above-described embodiment one reaction system, healthy plant, PNRSV, CMV, PVX, PVY, BBWV are detected simultaneously, the specificity of checking R T-LAMP method.Take the healthy plant that extracts, infected and extracted to such an extent that RNA carries out RT-LAMP amplified reaction as detecting template in the plant leaf of PNRSV, CMV, PVX, PVY, BBWV, only in PNRSV reaction tubes, there is positive reaction, other reaction tubess are all negative.This detection system of presentation of results high specificity, can detect PNRSV specifically, with other viral no cross reaction.
Embodiment tetra-: the RT-LAMP Analysis of test results of PNRSV
The RNA extracting in PNRSV positive of take is template, detects respectively, with the negative contrast of RNA of extracting in health plant blade with this law and conventional RT-PCR.The PNRSV positive that occurs white precipitate after RT-LAMP amplification, simultaneously with the CP gene of RT-PCR amplification PNRSV, amplified production carries out agarose electrophoresis detection, the specific band of (460bp) occurs conforming to target CP gene fragment size, negative control occurs without band, conforms to expected results.Proof RT-LAMP can detect the PNRSV in sample, consistent with the detected result of RT-PCR.
Claims (2)
1. a reverse transcription loop-mediated isothermal amplification detection method of setting up Prunus necrotic ring spot virus, is characterized in that, concrete detection method step is as follows:
(1) be designed for detect Prunus necrotic ring spot virus LAMP primer: according to the CP gene conserved sequence of the CP gene order of the PNRSV isolate of announcing in prior art report and GenBank, utilize LAMP primer-design software Primer Explorer V4 for 6 zone design, the 1 cover Auele Specific Primer of PNRSV isolate CP gene conserved sequence, comprise 1 pair of outer primer and 1 pair of inner primer, primer sequence is as follows:
F3：5′-AATCATACCCACGCTGGTG-3′
B3：5′-AATTCGGGGAGGCACATTC-3′
FIP：5′-TTCGCAGCCCTTTGTTGAGCC-TTGCAAGAAGTGCCATCCG-3′
BIP:5 '-TAGGGTTTCGAGCGGTGTAGGA-TCACGGTCCAAGTGGTCT-3 '; (2) extraction of the total RNA of plant: get 0.1g fresh plant blade or young shoot is ground to powder in liquid nitrogen, adopt RNAplant plus Reagen test kit to extract the total RNA of sample, add DEPC-ddH by extracting RNA product
2o dissolves, and-70 ℃ save backup;
(3) reverse transcription: the total RNA of plant extracting of take is template, sets up reverse transcription system, and cDNA sequence, the reverse transcription system of 20 μ L are synthesized in reverse transcription; Described reverse transcription system is RNA template 2 μ L, the dNTP1 μ L of 10mmol/L, the OligodT1 μ L of 50 μ mol/L, DEPC-ddH
2o10 μ L, processes 5min at 65 ℃, places 5min on ice, adds 5 * first-strand Buffer4 μ L, the M-MLV ThermoScript II 1 μ L of 200U/ μ L, the RNA enzyme inhibitors 1 μ L of 40U/ μ L; Reaction conditions is placed 10min at 30 ℃, 42 ℃ of insulation 1h, then in 95 ℃ of reaction 5min;
(4) preparation LAMP reaction system: described LAMP reaction system is, reverse transcription product cDNA5.0uL, the dNTP6.0uL of 2.5mmol/L, the primers F 3 of 10 μ mol/L, each 0.5uL of B3, each 4.0uL of the primers F IP of 10 μ mol/L, BIP, the Betain5.0uL of 100 μ mol/L, the MgSO of 100mmol/L
41.5uL, the Bst archaeal dna polymerase 1.0uL of 8U/ μ L arranges negative control simultaneously, and reaction system is that the consumption of each component of 27uL is: 10 * Buffer damping fluid 2.5uL;
(5) by LAMP response procedures, reaction template is increased; Described LAMP response procedures: 95 ℃ of heating 5min; Chilling 3min on ice, then add 1.0 μ LBst archaeal dna polymerases, 65 ℃ of water-bath 1.5h, 80 ℃ of deactivation 2min;
(6) by above-mentioned steps, increase and finish the variation of rear visual inspection reaction tubes turbidity, carry out the detection of LAMP amplified production.
2. the reverse transcription loop-mediated isothermal amplification detection method of setting up Prunus necrotic ring spot virus as claimed in claim 1, it is characterized in that, the variation of the described reaction tubes turbidity showing according to reaction mixture judges detected result, finishes the variation of rear visual inspection reaction tubes turbidity by amplification; To at the bottom of observation tube after amplified production high speed centrifugation, have or not magnesium pyrophosphate white precipitate, as occurred, white precipitate judges that detected result is positive, in sample, infect and have Prunus necrotic ring spot virus, on the contrary negative.
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